The present invention is directed to a method for the production of ribavirin and related compounds. The systematic name for ribavirin is 1-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide. Compounds of this type are known antiviral agents. Reference is made to U.S. Pat. No. 3,798,209 of Joseph T. Witkowski et al issued Mar. 19, 1974. Throughout the present specification, reference will be made to ribavirin. It will be understood that related compounds having a ribose group attached to a triazole are also intended.
There are several methods for the production of ribavirin. Chemical methods (those methods not using enzymes) are expensive. Expensive starting materials and process steps characterized by low yields are common.
As a result, bioconversion methods have been extensively studied. In these methods, an enzyme or enzymes are used to attach the ribose to the triazole. In some cases, the enzyme is first isolated and then used as the catalyst. Reference is made to U.S. Pat. No. 3,976,545 of Witkowski et al issued Aug. 24, 1976. In this patent there is disclosed a method wherein the ribose donor is ribose-1-phosphate. The triazole acceptor 1,2,4-triazole-3-carboxamide is reacted with the donor in the presence of nucleoside phosphorylase at a temperature between 0.degree. C. and 50.degree. C. The source of the enzyme is broadly disclosed.
Methods involving fermentation are also well known. In these methods the 1,2,4-triazole-3-carboxamide is added to a culture medium containing proliferating microorganisms such as a microorganism from the genus Brevibacterium. In this case, the necessary ribose donor comes from the fermentation medium as the organisms grow. Since the organisms are growing, the temperature is relatively low. Typical temperatures are between 20.degree. C. and 40.degree. C. Reaction times are very long, typically on the order of days.
In U.S. Pat. No. 4,458,016 to Yamanaka et al issued July 3, 1984 there is described a method that is very similar to the method of the U.S. Pat. No. 3,976,545 discussed above except that the temperature is between 55.degree. and 65.degree. C. Rather than isolated enzyme, whole cells containing the necessary activity can be used. Comparative results in the specification of this patent with the specific materials used indicate that the amount of ribavirin that is produced is very low at 70.degree. C. and negligible at 75.degree. C. and 80.degree. C. (see table 3 at column 7) The microorganism that was used in this test was Klebsiella pneuminiae and the ribose donor was either ribose-1-phosphate or uridine.
In U.S. Pat. No. 4,614,719 to Fujishima et al issued Sept. 30, 1986 there is disclosed a method that is similar to the method of U.S. Pat. No. 4,458,016 discussed above. In U.S. Pat. No. 4,614,719 a Brevibacterium acetylicum microorganism is used under nonproliferative conditions. A wide variety of ribose donors can be used according to this disclosure and the temperature can be between 40.degree. C. and 80.degree. C. However, in the examples, inosine is predominantly used as the donor and the temperature is usually 60.degree. C. In example 2 a variety of donors are tested with the B. acetylicum at 60.degree. C. and in Example 4 a variety of temperatures up to 70.degree. C. are tested with that microorganism and inosine as the donor. The yield of ribavirin dramatically decreases between 65.degree. and 70.degree. C.
The bioconversion method of U.S. Pat. No. 4,458,016 and U.S. Pats. No. 4,614,719 offer many advantages over the previous chemical method and the methods where the enzyme had to be isolated before use. However, additional improvements were still needed. For example, these methods produce ribavirin at a rate that is slower than desired. In U.S. Pat. No. 4,619,714 examples, the typical reaction time is 20 or 24 hours and the amount of ribavirin produced is relatively low. (Calculated to be at most about 10 g/L based on the data given.) Thus, while the % yield (actually the % conversion as described below) might be acceptable in these methods, the productivity of these methods is less than desired. Further, since the ribavirin is produced in only dilute solution, the recovery is more expensive than desired.